I. Field of the Invention
The present invention relates to novel methods, compositions and kits for amplifying, detecting and quantifying one or multiple target nucleic acid sequences in a sample, including target sequences that differ by as little as one nucleotide. The invention has broad applicability for research, environmental and genetic screening, and diagnostic applications, such as for detecting and quantifying sequences that indicate the presence of a pathogen, the presence of a gene or an allele, or the presence of a single nucleotide polymorphism (SNP) or other type of gene mutation or variant. The invention also relates to novel methods, compositions and kits for detecting and quantifying a broad range of analytes by detecting a target sequence that is joined to an analyte-binding substance.
II. Description of Related Art
Transcription of DNA into mRNA is regulated by the promoter region of the DNA. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA, and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA. RNA polymerases from different species typically recognize promoter regions comprised of different sequences. In order to obtain a transcription product by in vitro or in vivo transcription, the promoter driving transcription of the gene or DNA sequence must be a cognate promoter for the RNA polymerase, meaning that it is recognized by the RNA polymerase.
There are a number of methods in the art for detecting nucleic acid sequences, including point mutations. The presence of a nucleic acid sequence can indicate, for example, the presence of a pathogen, or the presence of particular genes or mutations in particular genes that correlate with or that are indicative of the presence or status of a disease state, such as, but not limited to, a cancer.
Examples of methods that involve in vitro transcription for making probes are described in: Murakawa et al., DNA 7:287-295, 1988; Phillips and Eberwine, Methods in Enzymol. Suppl. 10:283-288, 1996; Ginsberg et al., Ann. Neurol. 45:174-181, 1999; Ginsberg et al., Ann. Neurol. 48:77-87, 2000; VanGelder et al., Proc. Natl. Acad. Sci. USA 87:1663-1667, 1990; Eberwine et al., Proc. Natl. Acad. Sci. USA 89:3010-3014, 1992; U.S. Pat. Nos. 5,021,335; 5,168,038; 5,545,522; 5,514,545; 5,716,785; 5,891,636; 5,958,688; 26,291,170; and PCT Patent Applications WO 00/75356 and WO 02/065093.
Still other methods use in vitro transcription as part of a process for amplifying and detecting one or more target nucleic acid sequences in order to detect the presence of a pathogen, such as a viral or microbial pathogen, that is a causative agent for a disease or to detect a gene sequence that is related to a disease or the status of a disease for medical purposes. Examples where in vitro transcription methods have been used for medical purposes include U.S. Pat. Nos. 5,130,238; 5,194,370; 5,399,491; 5,409,818; 5,437,990; 5,466,586; 5,554,517; 5,665,545; 6,063,603; 6,090,591; 6,100,024; and 6,410,276; Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173, 1989; Fahy et al, In: PCR Methods and Applications, pp. 25-33, 1991; PCT Patent Application Nos. WO 89/06700 and WO 91/18155; and European Patent Application Nos. 0427073 A2 and 0427074 A2.
Still other methods detect sequences or mutations using methods that involve ligation of adjacently hybridizing oligonucleotide probes or ligation of non-adjacently hybridizing probes following a process such as primer extension. Ligation detection methods include those disclosed in European Patent Application Publication Nos. 0246864 A2 and 0246864 B1 of Carr; U.S. Pat. Nos. 4,883,750; 5,242,794; 5,521,065; 5,962,223; and 6,054,266 of Whiteley, N. M. et al.; U.S. Pat. No. 4,988,617 of Landegren and Hood; U.S. Pat. No. 5,871,921 of Landegren and Kwiatkowski; U.S. Pat. No. 5,866,337 of Schon; European Patent Application Publication Nos. 0320308 A2 and 0320308 B1 of Backman and Wang; PCT Publication No. WO 89/09835 of Orgel and Watt and European Patent Publication No. 0336731 B1 of Bruce Wallace; U.S. Pat. No. 5,686,272 of Marshall et al.; U.S. Pat. No. 5,869,252 of Bouma et al.; U.S. Pat. Nos. 5,494,810; 5,830,711; 6,054,564; 6,027,889; 6,268,148; and 6,312,892 of Barany et al.; U.S. Pat. Nos. 5,912,148 and 6,130,073 of F. Eggerding; U.S. Pat. No. 6,245,505 B1 of Todd and Fuery; European Patent Application Publication No. 0357336 A2 of Ullman et al.; U.S. Pat. No. 5,427,930 of Birkenmeyer et al. and U.S. Pat. No. 5,792,607 and European Patent Publication Nos. 0439182 A2 and EP 0439182 B1 of Backman et al.; U.S. Pat. Nos. 5,679,524; and 5,952,174 of Nikifoorov et al.; U.S. Pat. No. 6,025,139 of Yager and Dunn; and U.S. Pat. No. 6,355,431 B1 of Chee and Gunderson.
In addition, U.S. Pat. No. 6,153,384 of Lynch et al. discloses an assay to identify ligase activity modulators by ligation of a labeled nucleic acid to an immobilized capture nucleic acid in the presence of a potential ligase activity modulator. Furthermore, Mahajan et al., disclose in U.S. Pat. No. 5,976,806 a quantitative and functional DNA ligase assay that uses a linearized plasmid containing a reporter gene, wherein ligase activity is followed by the extent of coupled transcription-translation of the reporter gene.
Also, in U.S. Pat. No. 5,807,674 Tyagi discloses detection of RNA target sequences by ligation of the RNA binary probes, wherein a substrate for Q-beta replicase is generated.
In PCT Patent Application No. WO 92/01813, Ruth and Driver disclosed a process for synthesizing circular single-stranded nucleic acids by hybridizing a linear polynucleotide to a complementary oligonucleotide and then ligating the linear polynucleotide. They further disclosed a process for generating multiple linear complements of the circular single-stranded nucleic acid template by extending a primer more than once around the circular template using a DNA polymerase.
Japanese Patent Nos. JP4304900 and JP4262799 of Toshiya et al., disclose detection of a target sequence by ligation of a linear single-stranded probe having target-complementary 3′- and 5′-end sequences which are adjacent when the linear probe is annealed to a target sequence in the sample, followed by either rolling circle replication or in vitro transcription of the circular single-stranded template. Toshiya et al., disclose that in vitro transcription is performed by first annealing to the circular single-stranded template a complementary nucleotide primer having an anti-promoter sequence in order to form a double-stranded promoter, and then transcribing the circular single-stranded template having the annealed anti-promoter primer with an RNA polymerase that has helicase-like activity, such as T7, T3 or SP6 RNA polymerase.
In U.S. Pat. Nos. 6,344,329; 6,210,884; 6,183,960; 5,854,033; 6,329,150; 6,143,495; 6,316,229; and 6,287,824, Paul M. Lizardi also disclose the use of rolling circle replication to amplify and detect nucleic acid sequences. Lizardi further describes use of RNA polymerase protopromoters in the circular probe so that tandem-sequence single-stranded protopromoter-containing DNA products resulting from rolling circle replication can be transcribed by a cognate T7-type RNA polymerase following conversion of said DNA products to a form containing double-stranded promoters.
Furthermore, Kool et al., have disclosed synthesis of DNA or RNA multimers, meaning multiple copies of an oligomer or oligonucleotide joined end to end (i.e., in tandem) by rolling circle replication or rolling circle transcription, respectively, of a circular DNA template molecule. Rolling circle replication uses a primer and a strand-displacing DNA polymerase, such as phi 29 DNA polymerase. With respect to rolling circle transcription, it was shown these circular single-stranded DNA (ssDNA) molecules can be efficiently transcribed by phage and bacterial RNA polymerases (Prakash, G. and Kool, E., J. Am. Chem. Soc. 114: 3523-3527, 1992; Daubendiek, S. L. et al., J. Am. Chem. Soc. 117: 7818-7819, 1995; Liu, D. et al., J. Am. Chem. Soc. 118: 1587-1594, 1996; Daubendiek, S. L. and Kool, E. T., Nature Biotechnol., 15: 273-277, 1997; Diegelman, A. M. and Kool, E. T., Nucleic Acids Res., 26: 3235-3241, 1998; Diegelman, A. M. and Kool, E. T., Chem. Biol., 6: 569-576, 1999; Diegelman, A. M. et al., BioTechniques 25: 754-758, 1998; Frieden, M. et al., Angew. Chem. Int. Ed. Engl. 38: 3654-3657, 1999; Kool, E. T., Acc. Chem. Res., 31: 502-510, 1998; U.S. Pat. Nos. 5,426,180; 5,674,683; 5,714,320; 5,683,874; 5,872,105; 6,077,668; 6,096,880; and 6,368,802). Rolling circle transcription of these circular ssDNAs occurs in the absence of primers, in the absence of a canonical promoter sequence, and in the absence of any duplex DNA structure, and results in synthesis of linear multimeric complementary copies of the circle sequence up to thousands of nucleotides in length. Transcription of the linear precursor of the circular ssDNA template yielded only a small amount of RNA transcript product that was shorter than the template.
Fire and Xu (U.S. Pat. No. 5,648,245; Fire, A. and Xu, S-Q, Proc. Natl. Acad. Sci. USA, 92: 4641-4645, 1995) also disclose methods for using rolling circle replication of small DNA circles to construct oligomer concatamers.
Other researchers, including, but not limited to, Mahtani (U.S. Pat. No. 6,221,603), Rothberg et al., (U.S. Pat. No. 6,274,320), Dean et al., (Genome Res., 11: 1095-1099, 2001), Lasken et al., (U.S. Pat. No. 6,323,009), and Nilsson et al., (Nucleic Acids Res., 30 (14): e66, 2002) disclose other methods and applications of rolling circle amplification. Also, Pickering et al. (Nucleic Acids Res., 30 (12): e60, 2002) discloses a ligation and rolling circle amplification method for homogeneous end-point detection of single nucleotide polymorphisms (SNPs).
Although a number of nucleic acid amplification methods have been described in the art, there is a continuing need for methods and assays for detecting nucleic acids that are specific and accurate, yet are easier and faster than current methods. The present invention provides novel assays, methods, compositions and kits that are simple in format and very rapid to perform, but that can be used to detect and quantify any of a broad range of analytes with a high degree of specificity and sensitivity, including both nucleic acid analytes and non-nucleic acid analytes. With respect to analytes comprising a target nucleic acid, the invention provides assays, methods and kits that can detect and distinguish between target sequences, including sequences that differ even by only a single nucleotide, such as for analysis of single nucleotide polymorphisms.
All of the methods above for amplifying and detecting one or more target nucleic acid sequences use a double-stranded transcription promoter. In contrast to the methods in the art, the present invention provides methods, compositions and kits for detecting target nucleic acid sequences using an RNA polymerase that uses single-stranded DNA promoters and templates and that lacks helicase-like activity, as well as other advantages and benefits that will be clear from reading the specification below.